Bioprospecting of lignocellulolytic microorganisms for management of agricultural residues

Abstract

The study entitled “Bioprospecting of lignocellulolytic microorganisms for management of agricultural residues” was conducted at College of Agriculture, Vellayani during 2023 – 2024. The objective of the study was to isolate, screen and characterize microorganisms with lignocellulolytic properties for management of agricultural residues. A total of 43 bacterial isolates were isolated from decaying wood, spent mushroom substrate, saw dust, and garden soil rich in decaying plant matter by serial dilution and plating on Hans carboxymethyl cellulose (CMC) medium. These isolates were screened for cellulolytic activity on Hans CMC medium. Cellulolytic index was estimated for 17 isolates with measurable clearance zones around their colonies indicating cellulolytic activity. Isolates with cellulolytic index above 2.0 were selected for further analysis. Among the isolates thus selected, isolate CLSM02 recorded the highest cellulolytic index of 5.83. All the selected isolates except CLDW10 demonstrated growth on lignin basal medium indicating potential ligninolytic activity. The cellulase activity of all the selected isolates was estimated by DNS method using CMC and filter paper as substrates. The bacterial isolate CLSD07 exhibited highest cellulase activity of 8.49 IU mL-1 min-1 when CMC was given as substrate. Among the isolates tested, only CLDW10, CLSG06, CLSD04, and CLSM02 were capable of degrading filter paper. The isolate CLSD04 recorded the highest filter paper cellulase (Fpase) activity of 2.50 IU mL-1 min-1. Estimation of ligninolytic enzymes, viz., laccase, lignin peroxidase (LiP) and manganese peroxidase (MnP) was done using substrates 2, 2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) (ABTS), pyrogallol and guaiacol respectively. The bacterial isolate CLSM03 recorded highest laccase activity of 1.7 U mL-1. The highest LiP activity was observed in CLSD08 (15.49 U mL-1), which was on par with CLDW04 (15.37 U mL-1) on 72h after incubation. Three isolates exhibited 80 manganese peroxidase activity. The bacterial isolate CLSM03 recorded highest manganese peroxidase activity of 2.27 U mL-1. Based on their enzyme activity profiles, the best five isolates, CLSD04, CLSD07, CLSD08, CLSM02, and CLSM03, were selected for further analyses. All the selected isolates were gram positive, rod shaped and endospore formers. All the bacterial isolates were identified as Bacillus sp. by morphological and biochemical characterization. Molecular identification by 16S rRNA gene sequencing revealed that the isolates CLSD04 were Calidifontibacillus erzurumensis, CLSD07 were Bacillus velezensis, CLSD08 were Bacillus haynesii, CLSM02 were Bacillus stercoris and CLSM03 were Bacillus licheniformis respectively. The presence of cellulase and laccase genes was confirmed by gene-specific amplification. On PCR amplification with specific primer for cellulase, all five isolates exhibited bands corresponding to the cellulase gene. Bands corresponding to the laccase gene was detected in CLSM02 and CLSM03 on PCR amplification with primer for laccase. The selected isolates viz., Calidifontibacillus erzurumensis CLSD04, Bacillus velezensis CLSD07, Bacillus haynesii CLSD08, Bacillus stercoris CLSM02, and Bacillus licheniformis CLSM03, were found to be compatible with each other. A consortium of the compatible isolates was prepared. The efficacy of the lignocellulolytic consortium to degrade raw coir pith was assessed. The prepared consortium was sprayed on to thoroughly washed and dried raw coir pith and incubated for 15, 30 and 45 days. Tomato var. Anagha was raised in pro trays with coir pith subjected to various treatments as substrate. The highest plant height was recorded for T2 (raw coir pith sprayed with consortium and incubated for 30 days) at 19.30 ± 0.85 cm, followed by T5 (raw coir pith sprayed with water and incubated for 30 days) at 14.20 ± 0.31 cm. The treatments T2 and T5 exhibited the maximum dry weight (31.53 ± 0.98 mg and 28.84 ± 5.23 mg, respectively). The lignocellulolytic bacterial consortium consisting Calidifontibacillus erzurumensis CLSD04, Bacillus velezensis CLSD07, Bacillus haynesii CLSD08, Bacillus stercoris CLSM02, and Bacillus licheniformis CLSM03, exhibited 81 degradation of raw coir pith and plant growth promotion in tomato, suggesting its potential for agricultural residue management.